Dye binding assay reveals doxorubicin preference for DNA versus cardiolipin.

Colin A Fox, Robert O Ryan

Analytical biochemistry 2020 Apr 01

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Doxorubicin (DOX) is a potent anticancer agent that binds both DNA and cardiolipin (CL). To investigate DOX binding to CL versus DNA, aqueous soluble, CL-enriched nanoparticles, termed nanodisks (ND), were employed. Upon incubation with CL-ND, but not with phosphatidylcholine ND, DOX binding was detected. DOX binding to CL-ND was sensitive to buffer pH and ionic strength. To investigate if a DOX binding preference for DNA versus CL-ND exists, an agarose gel-based dye binding assay was developed. Under conditions wherein the commercial fluorescent dye, GelRed, detects a 636 bp DNA template following electrophoresis, DOX staining failed to visualize this DNA band. Incubation of the template DNA with DOX prior to electrophoresis resulted in a DOX concentration-dependent attenuation of GelRed staining intensity. When the template DNA was pre-incubated with equivalent amounts of free DOX or DOX-CL-ND, no differences in the extent of GelRed staining intensity attenuation were noted. When DOX was incubated with DNA alone, or a mixture of DNA and CL-ND, the extent of DOX-induced GelRed staining intensity attenuation was equivalent. Thus, DOX has a binding preference for DNA versus CL and, moreover, DOX-CL-ND offer a potential strategy to prevent DOX-induced cardiotoxicity while not affecting its affinity for DNA. Copyright © 2020 Elsevier Inc. All rights reserved.

Citation

Colin A Fox, Robert O Ryan. Dye binding assay reveals doxorubicin preference for DNA versus cardiolipin. Analytical biochemistry. 2020 Apr 01;594:113617